Identification of Host Genes for Retroviral Replication

描述（由申请人提供）：对模式生物酿酒酵母中的细胞周期、核转运、RNA加工、染色质结构、细胞骨架和囊泡运输的研究为后生动物中类似系统的表征提供了范例。我们和其他人已经在S.酿酒酵母作为逆转录病毒的模型。我们最近使用高通量筛选，以确定大约300个基因，其表达影响逆转录转Ty3，酵母逆转录病毒样元件。这些基因包括与DNA修复、RNA加工、蛋白质折叠、脂质代谢和囊泡运输相关的基因。在酵母系统中，我们正在相当详细地研究少数这些基因的作用机制。在这里，我们建议调查一个子集的19个基因复制的一个简单的哺乳动物逆转录病毒，莫洛尼鼠白血病病毒的影响。 本申请的目的是：1）靶基因功能将被shRNA破坏。候选基因的选择将基于几个标准，包括非必需小鼠同源物的存在、缺陷的严重性和Ty1转座的可能缺陷，Ty1是一种与Ty3比逆转录病毒更远相关的逆转录转座子。这些基因的活性将被shRNA破坏。2)将使用转染和感染试验确定对逆转录病毒感染性和生产的影响。3)将确定负作用基因过表达的影响。筛选中鉴定的一些基因对Ty3转座起负作用。这些基因的过度表达是否会降低逆转录病毒的复制将被确定。4)将确定逆转录病毒复制受有效候选基因影响的生化点。对于确定对逆转录病毒复制的shRNA或过表达的影响的基因，将测定复制中的蛋白质、RNA和DNA中间体，以确定候选基因影响复制的生命周期阶段。虽然影响可能是直接或间接的，但对基因功能的了解以及生命周期中的影响点，应该对影响机制提供重要的见解。这种方法具有很高的潜力，在逆转录病毒研究和确定新的治疗靶点开辟意想不到的途径。

DESCRIPTION (provided by applicant): Studies of the cell cycle, nuclear transport, RNA processing, chromatin structure, the cytoskeleton, and vesicular trafficking in the model organism Saccharomyces cerevisiae have provided paradigms for characterization of analogous systems in metazoans. We and others have characterized Ty retrotransposons in S. cerevisiae as models of retroviruses. We have recently used high throughput screening to identify roughly 300 genes, expression of which affects retrotransposition of Ty3, a yeast retrovirus-like element. These genes include ones related to DNA repair, RNA processing, protein folding, lipid metabolism, and vesicular trafficking. In the yeast system, we are studying the mechanism of action of a small number of these genes in considerable detail. Here we propose to survey a subset of nineteen genes for effects on replication of a simple mammalian retrovirus, Moloney murine leukemia virus. The goals of this application are: 1) Target gene function will be disrupted by shRNA. Candidate genes will be chosen based on several criteria, including existence of a nonessential mouse homolog, severity of defect, and possible defects for transposition of Ty1 a retrotransposon more distantly related to Ty3 than retroviruses. Activity of these genes will be disrupted by shRNA. 2) Effects on retrovirus infectivity and production will be determined using transfection and infection assays. 3) The effect of overexpression of negatively acting genes will be determined. Some genes identified in the screen act negatively on Ty3 transposition. Whether overexpression of these genes decreases retrovirus replication will be determined. 4) The biochemical point at which retrovirus replication is affected by effective candidate genes will be determined. For genes where an effect on retrovirus replication of shRNA or overexpression is determined, protein, RNA and DNA intermediates in replication will be assayed in order to identify the stage in the lifecycle where replication is impacted by the candidate gene. Although effects could be direct or indirect, knowledge of the function of the gene together with the point of effect in the lifecycle, should offer significant insight as to the mechanism of the effect. This approach has high potential for opening unexpected avenues in retrovirus research and identifying novel targets for therapeutics.